Materials 2011, 4, 327-338; doi:10.3390/ma4010327 OPEN ACCESS
materials ISSN 1996-1944 www.mdpi.com/journal/materials Article
Cell-Based Fabrication of Organic/Inorganic Composite Gel Material Takuya Matsumoto 1,*, Ami Mizuno 1, Miki Kashiwagi 1, Shin-suke Yoshida 1, Jun-ichi Sasaki 1 and Takayoshi Nakano 2 1
2
Department of Oromaxillofacial Regeneration, Osaka University, 1-8 Yamada-oka, Suita, Osaka 565-0871, Japan Division of Materials & Manufacturing Science, Osaka University, Suita, Osaka 565-0871, Japan
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +81-6-6879-2919; Fax: +81-6-6879-2919. Received: 31 December 2010 / Accepted: 21 January 2011 / Published: 24 January 2011
Abstract: Biomaterials containing components similar to the native biological tissue would have benefits as an implantable scaffold material. To obtain such biomimetic materials, cells may be great contributors because of their crucial roles in synthetic organics. In addition, the synthesized organics—especially those derived from osteogenic differentiated cells—become a place where mineral crystals nucleate and grow even in vitro. Therefore, to fabricate an organic/inorganic composite material, which is similar to the biological osteoid tissue, bone marrow derived mesenchymal stem cells (BMSCs) were cultured in a 3D fibrin gel in this study. BMSCs secreted bone-related proteins that enhanced the biomineralization within the gel when the cells were cultured with an osteogenic differentiation medium. The compositions of both synthesized matrices and precipitated minerals in the obtained materials altered depending on the cell culture period. The mineral obtained in the 3D gel showed low crystalline hydroxyapatite. The composite materials also showed excellent osteoconductivity with new bone formation when implanted in mice tibiae. Thus, we demonstrated the contributions of cells for fabricating implantable organic/inorganic composite gel materials and a method for controlling the material composition in the gel. This cell-based material fabrication method would be a novel method to fabricate organic/inorganic composite biomimetic materials for bone tissue engineering. Keywords: hydrogel; organic/inorganic composite; 3D; biomimetic material
Materials 2011, 4
328
1. Introduction Bone tissue has critical roles in supporting body weight, enabling motility, and protecting important organs [1,2]. Bone tissue also works as a reservoir for inorganic ions, such as calcium and phosphorus, and supplies a place for hematopoiesis to occur [3]. For bone defects caused by regional segmentation for tumor extractions, necrosis due to X-ray radiation, or injury [4,5], the bones are reconstructed using biomaterials, including titanium or sintered ceramic materials, to recover the bone functions [6,7]. This bone reconstruction supports body motility and rebuilds the morphology of the original tissue. However, metals or sintered ceramic biomaterials have limited abilities for generating more complicated bone functions such as supplying calcium and phosphorus or hematopoiesis. Therefore, bone tissue regeneration by combining biomaterials and biologic materials (e.g., cells and soluble factors) has been investigated in the past few decades [8-10]. For example, proteins, sugars, and bioceramics have been evaluated as functional carriers for cells and/or soluble factors and have significantly enhanced bone regeneration [11,12]. However, these biomaterials still have limited approval for clinical use because the natural polymers used in these biomaterials (e.g., type I collagen, hyaluronic acid) are often derived from domesticated animals. Therefore, biomaterials derived from autologous sources alone would have tremendous advantages. On the other hand, integrating implanted biomaterials with host hard tissues is also considered a challenging issue in bone tissue engineering [13]. For this integration, fabrication of implantable materials that contain both organics and minerals similar to the components of biological osteoid tissue might be effective, because naturally derived organics generally have higher affinity to host cell and tissue. However, native bony tissue contains the huge kinds of organic molecules derived from cell addition to apatite minerals. Hence, culturing cells in a three-dimensional (3D) hydrogel material in vitro would be an effective approach to fabricate biomimetic osteoid-like materials, because cells themselves secret osteoid proteins that enhance the mineral precipitations. Fibrin is a fibrous protein that forms fibrin clots to prevent further bleeding from an injured blood vessel. Because fibrinogen and thrombin, the source of fibrin, can be obtained from the peripheral blood of any individual [14], fibrin gel is one of the ideal biomaterials for tissue engineering purpose when applied to one’s self. Bone marrow derived mesenchymal stem cells (BMSCs) can differentiate into osteoblasts and secret a number of bone-related matrix proteins when they are cultured with a special osteogenic medium [15]. Moreover, the secreted proteins supply the location for mineral crystals to nucleate and grow [16]. Here, we hypothesized that culturing BMSCs in 3D fibrin gel would form organic/inorganic composite materials containing cells, bone matrix proteins, and minerals that are similar to biological osteoid tissue. Moreover, the matrix and mineral composition in the 3D constructs would be controlled by the cell culture conditions. To test these hypotheses, BMSCs were cultured in 3D fibrin gel for different culture periods (14–42 days) in floating culture condition. The chemical properties of the obtained 3D constructs were then investigated in this study.
Materials 2011, 4
329
2. Results and Discussion 2.1. BMSCs Culture in 3D Fibrin Gel Employing and culturing cells in 3D hydrogel would be a new method to synthesize organic/inorganic composite material that mimics biologically immature osteoid tissue. To address this goal, BMSCs were cultured in 3D fibrin gel with osteogenic differentiation medium. Fibrin gel containing cells that was formed in a silicone mold was initially cylinder-shaped but became spheroidal during the culturing (Figure 1). The gel size decreased dramatically when cells were contained in the gel (
materials ISSN 1996-1944 www.mdpi.com/journal/materials Article
Cell-Based Fabrication of Organic/Inorganic Composite Gel Material Takuya Matsumoto 1,*, Ami Mizuno 1, Miki Kashiwagi 1, Shin-suke Yoshida 1, Jun-ichi Sasaki 1 and Takayoshi Nakano 2 1
2
Department of Oromaxillofacial Regeneration, Osaka University, 1-8 Yamada-oka, Suita, Osaka 565-0871, Japan Division of Materials & Manufacturing Science, Osaka University, Suita, Osaka 565-0871, Japan
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +81-6-6879-2919; Fax: +81-6-6879-2919. Received: 31 December 2010 / Accepted: 21 January 2011 / Published: 24 January 2011
Abstract: Biomaterials containing components similar to the native biological tissue would have benefits as an implantable scaffold material. To obtain such biomimetic materials, cells may be great contributors because of their crucial roles in synthetic organics. In addition, the synthesized organics—especially those derived from osteogenic differentiated cells—become a place where mineral crystals nucleate and grow even in vitro. Therefore, to fabricate an organic/inorganic composite material, which is similar to the biological osteoid tissue, bone marrow derived mesenchymal stem cells (BMSCs) were cultured in a 3D fibrin gel in this study. BMSCs secreted bone-related proteins that enhanced the biomineralization within the gel when the cells were cultured with an osteogenic differentiation medium. The compositions of both synthesized matrices and precipitated minerals in the obtained materials altered depending on the cell culture period. The mineral obtained in the 3D gel showed low crystalline hydroxyapatite. The composite materials also showed excellent osteoconductivity with new bone formation when implanted in mice tibiae. Thus, we demonstrated the contributions of cells for fabricating implantable organic/inorganic composite gel materials and a method for controlling the material composition in the gel. This cell-based material fabrication method would be a novel method to fabricate organic/inorganic composite biomimetic materials for bone tissue engineering. Keywords: hydrogel; organic/inorganic composite; 3D; biomimetic material
Materials 2011, 4
328
1. Introduction Bone tissue has critical roles in supporting body weight, enabling motility, and protecting important organs [1,2]. Bone tissue also works as a reservoir for inorganic ions, such as calcium and phosphorus, and supplies a place for hematopoiesis to occur [3]. For bone defects caused by regional segmentation for tumor extractions, necrosis due to X-ray radiation, or injury [4,5], the bones are reconstructed using biomaterials, including titanium or sintered ceramic materials, to recover the bone functions [6,7]. This bone reconstruction supports body motility and rebuilds the morphology of the original tissue. However, metals or sintered ceramic biomaterials have limited abilities for generating more complicated bone functions such as supplying calcium and phosphorus or hematopoiesis. Therefore, bone tissue regeneration by combining biomaterials and biologic materials (e.g., cells and soluble factors) has been investigated in the past few decades [8-10]. For example, proteins, sugars, and bioceramics have been evaluated as functional carriers for cells and/or soluble factors and have significantly enhanced bone regeneration [11,12]. However, these biomaterials still have limited approval for clinical use because the natural polymers used in these biomaterials (e.g., type I collagen, hyaluronic acid) are often derived from domesticated animals. Therefore, biomaterials derived from autologous sources alone would have tremendous advantages. On the other hand, integrating implanted biomaterials with host hard tissues is also considered a challenging issue in bone tissue engineering [13]. For this integration, fabrication of implantable materials that contain both organics and minerals similar to the components of biological osteoid tissue might be effective, because naturally derived organics generally have higher affinity to host cell and tissue. However, native bony tissue contains the huge kinds of organic molecules derived from cell addition to apatite minerals. Hence, culturing cells in a three-dimensional (3D) hydrogel material in vitro would be an effective approach to fabricate biomimetic osteoid-like materials, because cells themselves secret osteoid proteins that enhance the mineral precipitations. Fibrin is a fibrous protein that forms fibrin clots to prevent further bleeding from an injured blood vessel. Because fibrinogen and thrombin, the source of fibrin, can be obtained from the peripheral blood of any individual [14], fibrin gel is one of the ideal biomaterials for tissue engineering purpose when applied to one’s self. Bone marrow derived mesenchymal stem cells (BMSCs) can differentiate into osteoblasts and secret a number of bone-related matrix proteins when they are cultured with a special osteogenic medium [15]. Moreover, the secreted proteins supply the location for mineral crystals to nucleate and grow [16]. Here, we hypothesized that culturing BMSCs in 3D fibrin gel would form organic/inorganic composite materials containing cells, bone matrix proteins, and minerals that are similar to biological osteoid tissue. Moreover, the matrix and mineral composition in the 3D constructs would be controlled by the cell culture conditions. To test these hypotheses, BMSCs were cultured in 3D fibrin gel for different culture periods (14–42 days) in floating culture condition. The chemical properties of the obtained 3D constructs were then investigated in this study.
Materials 2011, 4
329
2. Results and Discussion 2.1. BMSCs Culture in 3D Fibrin Gel Employing and culturing cells in 3D hydrogel would be a new method to synthesize organic/inorganic composite material that mimics biologically immature osteoid tissue. To address this goal, BMSCs were cultured in 3D fibrin gel with osteogenic differentiation medium. Fibrin gel containing cells that was formed in a silicone mold was initially cylinder-shaped but became spheroidal during the culturing (Figure 1). The gel size decreased dramatically when cells were contained in the gel (
